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Background Trilaciclib is an intravenous (IV) cyclin-dependent kinase 4/6 (CDK4/6) inhibitor. Preliminary data showed that adding trilaciclib prior to gemcitabine plus carboplatin (GCb) significantly increased overall survival (OS) compared with GCb alone among patients with metastatic triple-negative breast cancer (mTNBC) (Tan et al., Lancet Oncol. 2019;20:1587-1601). Here, final antitumor efficacy results (objective response rate [ORR], progression-free survival [PFS], and OS) are reported for the whole study population, and in cohorts according to CDK4/6 dependence and level of programmed death ligand-1 (PD-L1) expression. Methods This was a randomized, open-label, phase 2 study of patients with mTNBC who had received ≤2 previous lines of chemotherapy in the recurrent/metastatic setting (NCT02978716). Patients were randomized (1:1:1) to receive GCb on days 1 and 8 (group 1, n=34), trilaciclib prior to GCb on days 1 and 8 (group 2, n=33), or trilaciclib alone on days 1 and 8 and prior to GCb on days 2 and 9 (group 3, n=35), in 21-day cycles. PFS and OS (prespecified secondary endpoints) were assessed in the intention-to-treat (ITT) population, and ORR in response-evaluable patients. Patient tumors were characterized as CDK4/6 independent (basal-like) or indeterminate (HER2-enriched, normal-like, luminal A/B) according to the established PAM50 signature, or CDK4/6 dependent (luminal androgen receptor) or indeterminate (basal-like 1/2, mesenchymal) according to the established Lehmann signature. PD-L1 expression was scored as negative or positive if Results Median follow-up was 8.4 months (range: 0.1-25.7) for group 1, 14.0 months (1.3-33.6) for group 2, and 15.3 months (3.5-33.7) for group 3. The ORR among response-evaluable patients was 7/24 (29.2%) in group 1, 15/30 (50.0%) in group 2, and 12/31 (38.7%) in group 3. Median PFS (95% confidence interval [CI]) in the ITT population was 5.7 (3.3, 9.9) months in group 1, 9.4 (6.1, 11.9) months in group 2, and 7.3 (6.2, 13.9) months in group 3, with hazard ratios (HRs) of 0.62 (P = 0.2099) and 0.63 (P = 0.1816), for groups 2 and 3 versus group 1, respectively. Overall, 73.5%, 39.4%, and 57.1% of patients in groups 1, 2, and 3 had died. Median OS (95% CI) was 12.6 (6.3, 15.6) months in group 1, not reached (NR) (10.2, NR) in group 2 (HR = 0.31, P = 0.0016), and 17.8 (12.9, 32.7) months in group 3 (HR = 0.40, P = 0.0004). For groups 2 and 3 combined, median OS was 19.8 (14.0, NR) months (HR = 0.37, P Conclusions Mature data from this study confirm that administering trilaciclib prior to GCb enhances antitumor efficacy compared with GCb alone, with statistically significant improvements in OS. Subgroup analyses suggest that adding trilaciclib prior to GCb benefits patients regardless of CDK4/6 dependence status and PD-L1 expression. Additional immune subtyping analyses are ongoing and will be presented. Group 1Group 2Group 3PD-L1 +vePD-L1 –vePD-L1 +vePD-L1 –vePD-L1 +vePD-L1 –vePatients, n171016101616ORR, n (%)4 (23.5)3 (30.0)8 (50.0)4 (40.0)7 (43.8)4 (25.0)Median PFS, months (95% CI)3.5 (2.2, NR)9.5 (5.2, NR)7.9 (4.3, NR)11.9 (8.8, NR)9.0 (6.2, NR)6.9 (6.4, NR)P value (Wald Test)––0.3470.6040.0690.766HR (95% CI)––0.70 (0.3, 1.5)0.76 (0.3, 2.2)0.46 (0.2, 1.1)1.16 (0.4, 3.1)Median OS, months (95% CI)10.5 (6.3, 18.8)13.9 (12.6, NR)20.1 (10.2, NR)NR (9.4, NR)32.7 (15.3, NR)17.8 (12.9, NR)P value (Wald Test)––0.0280.0830.020.239HR (95% CI)––0.35 (0.1, 0.9)0.34 (0.1, 1.2)0.33 (0.1, 0.8)0.57 (0.2, 1.5)HR and P values are for comparisons between group 2 versus group 1, and group 3 versus group 1.+ve, positive; –ve, negative; CI, confidence interval; HR, hazard ratio; NR, not reached; ORR, objective response rate; OS, overall survival; PD-L1, programmed death ligand-1; PFS, progression-free survival. Citation Format: Joyce O'Shaughnessy, Gail S Wright, Anu R Thummala, Michael A Danso, Lazar Popovic, Timothy J Pluard, Hyo S Han, Željko Vojnović, Nikola Vasev, Ling Ma, Donald A Richards, Sharon T Wilks, Dušan Milenković, Jie Xiao, Jessica A Sorrentino, Janet Horton, Antoinette R Tan. Trilaciclib improves overall survival when given with gemcitabine/carboplatin in patients with metastatic triple-negative breast cancer: Final analysis of a randomized phase 2 trial [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD1-06.

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.

BACKGROUND AND AIM: Exposure to neurotoxic metals occurs as a mixture, yet most studies consider single exposures, potentially missing the combined effects of environmental metals. Further, health ...

Background Phthalate exposure has been associated with increased childhood behavioral problems. Existing studies failed to include phthalate replacements and did not account for high correlations among phthalates. Phthalates’ exposure is higher in Mexico than in U.S. locations, making it an ideal target population for this study. Aim To examine associations between 15 maternal prenatal phthalate metabolite concentrations and children's behavioral problems. Methods We quantified phthalate metabolites in maternal urine samples from maternal-child dyads (n = 514) enrolled in the Programming Research in Obesity, Growth Environment and Social Stress (PROGRESS) birth cohort in Mexico City. We performed least absolute shrinkage and selection operator (LASSO) regressions to identify associations between specific-gravity adjusted log2-transformed phthalate metabolites and parent-reported 4–6 year old behavior on the Behavior Assessment System for Children (BASC-2), accounting for metabolite correlations. We adjusted for socio-demographic and birth-related factors, and examined associations stratified by sex. Results Higher prenatal mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) urinary concentrations were associated with increased hyperactivity scores in the overall sample (β = 0.57, 95% CI = 0.17, 1.13) and in girls (β = 0.54, 95% CI = 0.16, 1.08), overall behavioral problems in boys (β = 0.58, 95% CI = 0.20, 1.15), and depression scores in boys (β = 0.44, 95% CI = 0.06, 0.88). Higher prenatal monobenzyl phthalate (MBzP) concentrations were associated with reduced hyperactivity scores in girls (s = −0.54, 95% CI = −1.08, −0.21). Discussion Our findings suggested that prenatal concentrations of phthalates and their replacements altered child neurodevelopment and those associations may be influenced sex.

Purpose Locoregional control for inflammatory breast cancers and chest wall recurrences is suboptimal, which has motivated interest in radiosensitization to intensify therapy. Preclinical studies have suggested a favorable therapeutic index when poly (ADP-ribose) polymerase inhibitors are used as radiosensitizers; clinical investigation is necessary to establish appropriate dosing and confirm safety. Patients and Methods We conducted a multi-institutional phase I study of veliparib and concurrent radiotherapy (RT) to the chest wall and regional lymph nodes in 30 patients with inflammatory or locally recurrent breast cancer after complete surgical resection. RT consisted of 50 Gy to the chest wall and regional lymph nodes plus a 10-Gy boost. A Bayesian time-to-event continual reassessment method escalated dose through four levels, with a 30% targeted rate of dose-limiting toxicity (DLT) measured during the 6 weeks of treatment plus 4 weeks of follow-up. DLTs were defined as confluent moist desquamation > 100 cm2, nonhematologic toxicity grade ≥ 3, toxicity that requires an RT dose delay > 1 week, absolute neutrophil count < 1,000/mm3, platelet count < 50,000/mm3, or hemoglobin < 8.0 g/dL if possibly, probably, or definitely related to study treatment. Results Five DLTs occurred: Four were moist desquamation (two each at 100 and 150 mg twice a day), and one was neutropenia (at 200 mg twice a day). The crude rate of any grade 3 toxicity (regardless of attribution) was 10% at year 1, 16.7% at year 2, and 46.7% at year 3. At year 3, six of 15 surviving patients had severe fibrosis in the treatment field. Conclusion Although severe acute toxicity did not exceed 30% even at the highest tested dose, nearly half of surviving patients demonstrated grade 3 adverse events at 3 years, which underscores the importance of long-term monitoring of toxicity in trials of radiosensitizing agents.

BackgroundTrilaciclib is an intravenous cyclin-dependent kinase 4/6 inhibitor approved to reduce the incidence of chemotherapy-induced myelosuppression in patients with extensive-stage small cell lung cancer (myeloprotection). In a randomized, open-label phase 2 trial in patients with metastatic triple-negative breast cancer (mTNBC), adding trilaciclib prior to gemcitabine/carboplatin (GCb) increased overall survival in both PD-L1–positive and –negative populations versus GCb alone.1 2 We investigated potential immune mechanisms of anti-tumor efficacy among patients who received trilaciclib plus GCb.MethodsPeripheral blood was collected prior to and on treatment for flow cytometric analysis, and total RNA isolated from diagnostic tumor biopsies for sequencing. Differential gene expression analysis between responders and non-responders was based on negative binomial distribution and related pathways identified by Kyoto Encyclopedia of Genes and Genomes pathway analysis. Tumor inflammation signatures and deconvolution-based approaches were used to assess the tumor immune microenvironment. PD-L1 expression was considered positive if ≥1% of the total tumor area contained PD-L1–labelled immune cells (Ventana SP142 assay). Patients were defined as responders (confirmed complete or partial response) or non-responders (stable or progressive disease) according to RECIST criteria.ResultsOf 68 patients who received trilaciclib prior to GCb, tumor response status and RNA sequencing data were available for 51 patients, comprising 24 responders and 27 non-responders. Tumors from responders had 253 differentially expressed genes compared with non-responders. Analysis of immune gene signatures revealed a higher T-cell exhaustion score at baseline among responders versus non-responders (P=0.044).Among patients with PD-L1–positive tumors, responders had a greater peripheral immune response at baseline compared with non-responders, including more T cells (P=0.037; particularly memory CD8 T cells [P=0.042]), and a trend toward fewer myeloid-derived suppressor cells (MDSCs). Additionally, tumors from responders had more dendritic cells (P=0.044) and a trend toward enriched tumor inflammation signatures compared with non-responders.By contrast, among patients with PD-L1–negative tumors, responders had similar peripheral immune populations at baseline compared with PD-L1–negative non-responders, but fewer MDSCs (P=0.016), and a trend toward increased T-cell numbers after two cycles of trilaciclib plus GCb.Responders with both PD-L1–positive and –negative tumors had increased numbers of naïve CD8 T cells after two treatment cycles compared with non-responders.ConclusionsThe data suggest that adding trilaciclib prior to GCb enhances antitumor efficacy by modulating the composition of immune cell subsets. The impact of trilaciclib on changes to the tumor-infiltrating immune response is being further investigated in a phase 3 trial in patients with mTNBC (NCT04799249).AcknowledgementsFlow cytometry and RNA sequencing analyses were performed by Covance, Inc., and Q2 Laboratory Solutions, respectively.Trial Registration www.clinicaltrials.govNCT02978716ReferencesTan AR, Wright GS, Thummala AR, Danso MA, Popovic L, Pluard TJ, Han HS, Vojnović Ž, Vasev N, Ma L, Richards DA, Wilks ST, Milenković D, Yang Z, Antal JM, Morris SR, O’Shaughnessy J. Trilaciclib plus chemotherapy versus chemotherapy alone in patients with metastatic triple-negative breast cancer: a multicentre, randomised, open-label, phase 2 trial. Lancet Oncol 2019;20(11):1587–1601.O’Shaughnessy J, Wright GS, Thummala AR, Danso MA, Popovic L, Pluard TJ, Han HS, Vojnović Ž, Vasev N, Ma L, Richards DA, Wilks ST, Milenković D, Xiao J, Sorrentino JA, Horton J, Tan AR. Abstract PD1-06: trilaciclib improves overall survival when given with gemcitabine/carboplatin in patients with metastatic triple-negative breast cancer: final analysis of a randomized phase 2 trial. Cancer Res 2021;81(4 Supplement):PD1-06.Ethics ApprovalThe study protocol and all associated amendments and study-related materials were approved by the institutional review board or independent ethics committee of each investigational site.

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) β. Pol β was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol β. Mitochondria from pol β-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol β-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol β and thus pol β plays a role in preserving mitochondrial genome stability.

Deciphering factors modulating DNA repair in chromatin is of great interest because nucleosomal positioning influences mutation rates. H3K56 acetylation (Ac) is implicated in chromatin landscape regulation, impacting genomic stability, yet the effect of H3K56Ac on DNA base excision repair (BER) remains unclear. We determined whether H3K56Ac plays a role in regulating AP site incision by AP endonuclease 1 (APE1), an early step in BER. Our in vitro studies of acetylated, well-positioned nucleosome core particles (H3K56Ac-601-NCPs) demonstrate APE1 strand incision is enhanced compared with that of unacetylated WT-601-NCPs. The high-mobility group box 1 protein enhances APE1 activity in WT-601-NCPs, but this effect is not observed in H3K56Ac-601-NCPs. Therefore, our results suggest APE1 activity on NCPs can be modulated by H3K56Ac.

Genomic DNA is susceptible to endogenous and environmental stresses that modify DNA structure and its coding potential. Correspondingly, cells have evolved intricate DNA repair systems to deter changes to their genetic material. Base excision DNA repair involves a number of enzymes and protein cofactors that hasten repair of damaged DNA bases. Recent advances have identified macromolecular complexes that assemble at the DNA lesion and mediate repair. The repair of base lesions generally requires five enzymatic activities: glycosylase, endonuclease, lyase, polymerase, and ligase. The protein cofactors and mechanisms for coordinating the sequential enzymatic steps of repair are being revealed through a range of experimental approaches. We discuss the enzymes and protein cofactors involved in eukaryotic base excision repair, emphasizing the challenge of integrating findings from multiple methodologies. The results provide an opportunity to assimilate biochemical findings with cell-based assays to uncover new insights into this deceptively complex repair pathway.

Maturational processes in the developing brain are disrupted by exposure to environmental toxicants, setting the stage for deviant developmental trajectories. Manganese (Mn) is an essential nutrient that is neurotoxic at high levels of exposure, particularly affecting the basal ganglia and prefrontal cortex. Both the intensity and timing of exposure matter; deciduous teeth can be used to retrospectively and objectively determine early-life windows of vulnerability. The aim of this pilot study was to examine associations between prenatal, early postnatal and childhood dentine Mn concentrations and intrinsic functional connectivity (iFC) of adolescents’ brains. 14 adolescents (12–18 years; 6 girls) from northern Italian regions with either current, historic or no Mn contamination, completed a 10-minute resting state functional Magnetic Resonance Imaging (MRI) scan in an 1.5T MRI scanner. We estimated prenatal, early postnatal and childhood Mn concentrations in deciduous teeth using laser ablation-inductively coupled plasma-mass spectrometry. We performed seed-based correlation analyses, focusing on six subcortical seeds (left and right caudate, putamen, pallidum) and one cortical seed (bilateral middle frontal gyrus) from Harvard-Oxford atlases. We examined linear and quadratic correlations between log-transformed Mn concentrations and seed-based iFC (Bonferroni-corrected p

Manganese is a naturally occurring metal that is a both an essential nutrient at lower levels and a neurotoxicant at higher levels. Recent studies suggest that higher prenatal manganese exposure re...

Objective:Examine associations between PBDE concentrations and functional connectivity of the reading network (RN) in a community sample.Method:Resting State fMRI data (rsfMRI) were acquired from 3...

Background Trilaciclib (T) is an IV cyclin dependent kinase (CDK) 4/6 inhibitor and first-in-class myelopreservation agent. Previous results from this phase II trial demonstrated that adding T to GC in mTNBC resulted in: 1) a longer duration and higher administered total dose of chemotherapy, 2) higher response rates and longer PFS, and 3) reduction in the frequency of some myelosuppression events. We now report preliminary OS and updated PFS.TableLBA22TableGroup 1 GC D1, 8 n=34Group 2 Trilaciclib + GC D1, 8 n=33Group 3 Trilaciclib D1, 2, 8, 9 + GC D2, 9 n=35Group 2 + Group 3 n=68Progression-free survival (PFS)Patients with events [n (%)]18 (52.9%)19 (57.6%)18 (51.4%)37 (54.4%)Median PFS (95% CI) [months]5.7 (3.4, 9.2)9.4 (6.1, 13.0)7.3 (6.2, 12.9)8.8 (6.4, 10.9)HR0.600.590.59p-value0.12560.11670.0628Overall survival (OS)Patients with death [n (%)]19 (55.9%)11 (33.3%)14 (40.0%)25 (36.8%)Median OS (95% CI) [months]12.6 (6.3, 15.6)20.1 (10.2, NE)17.8 (12.9, NE)20.1 (15.3, NE)HR0.370.390.40p-value0.04110.00440.0032 Methods This randomized, phase II, open-label study enrolled mTNBC patients with 0-2 prior chemotherapy regimens for recurrent/metastatic disease. Patients were randomized to GC alone on day 1, 8 (Group 1) or T + GC (Group 2) or T + GC (Group 3) using an alternative schedule (T D1, 2, 8, 9 + GC D2, 9) every 21 days until disease progression or unacceptable toxicity. The primary endpoint was reduction in GC-related neutropenia. PFS and OS were secondary endpoints. Results Among 102 enrolled patients, 98 were treated. 52% had ECOG PS 0 and 37.3% received prior chemotherapy for mTNBC. Median follow-up is 10.5 months; 3 patients remain on treatment. There have been no SAEs related to T. Drug exposure in Group 2/3 patients receiving T (median 168 days, 8 cycles) is longer than with GC alone (median 101 days, 4 cycles). PFS and OS in the intent to treat (ITT) population are summarized in the table below. Groups 2+3 (receiving T) had longer PFS compared with GC alone, HR of 0.59, p=0.0628, and OS was statistically improved with HR of 0.37 and 0.39 in Groups 2 and 3, p=0.0411 and p=0.0044, respectively. ORR were 36.7%, 50.0%, and 33.3% in Groups 3, 2, and 1, respectively. Conclusions The addition of T to GC resulted in a significant increase in OS in both T groups. Previously observed PFS benefit with T was also confirmed. Clinical trial identification NCT02978716. Legal entity responsible for the study G1 Therapeutics, Inc. Funding G1 Therapeutics, Inc. Disclosure J. O'Shaughnessy: Honoraria (self): AbbVie Inc; Honoraria (self): Agendia; Honoraria (self): Amgen Biotechnology; Honoraria (self): AstraZeneca; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Celgene Croporation; Honoraria (self): Eisai; Honoraria (self): Genentech; Honoraria (self): Genomic Health; Honoraria (self): GRAIL; Honoraria (self): Immunomedics; Honoraria (self): Heron Therapeutics; Honoraria (self): Ipsen Biopharmaceuticals; Honoraria (self): Jounce Therapeutics; Honoraria (self): Lilly; Honoraria (self): Merck; Honoraria (self): Myriad; Honoraria (self): Novartis; Honoraria (self): Ondonate Therapeutics; Honoraria (self): Pfizer. G.S. Wright: Honoraria (self): Total Health Conferencing; Shareholder / Stockholder / Stock options: Roche Holdings Ltd; Shareholder / Stockholder / Stock options: Odonate Therapeutics; Shareholder / Stockholder / Stock options: Puma Biotech; Research grant / Funding (institution), Shareholder / Stockholder / Stock options, PI does not receive direct research funding; institution received payment for conduct of trials: Macrogenics ; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: AbbVie; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Incyte; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Genentech; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Novartis; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Lilly; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Janssen; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Astellas; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Celgene; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: AstraZeneca; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Bristol-Myers Squibb; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Boehringer Ingelheim; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Medivation; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Merrimack; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Tesaro; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Pfizer. A. Thummala: Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Comprehensive Cancer Centers of Nevada. H.S. Han: Advisory / Consultancy: Lilly; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Novartis; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Pfizer; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Tesaro; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: TapImmune; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Seattle Genetics; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: BMS Prescient; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Horizon; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Karyopharm. S. Wilks: Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: US Oncology. J.A. Sorrentino: Shareholder / Stockholder / Stock options, Full / Part-time employment: G1 Therapeutics. Z. Yang: Shareholder / Stockholder / Stock options, Full / Part-time employment: G1 Therapeutics. J.K. Horton: Shareholder / Stockholder / Stock options, Full / Part-time employment: G1 Therapeutics; Honoraria (self), Research grant / Funding (self): Varian Medical Systems; Honoraria (self): Qfix; Leadership role: International Journal of Radiation Oncology, Biology, Physics; Leadership role: American Board of Radiology; Leadership role: ASTRO. A.R. Tan: Advisory / Consultancy: Celgene; Advisory / Consultancy: Immunomedics; Advisory / Consultancy, Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Genentech; Advisory / Consultancy: Novartis; Advisory / Consultancy, Research grant / Funding (self): Pfizer; Research grant / Funding (self): Merck; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: G1 Therapeutics; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Roche; Research grant / Funding (institution), PI does not receive direct research funding; institution received payment for conduct of trials: Tesaro; Leadership role, Breast Cancer Leader: Caris Precision Oncology Alliance. All other authors have declared no conflicts of interest.

The organophosphate insecticide chlorpyrifos (CPF), widely used for agricultural purposes, has been linked to neurodevelopmental deficits. Possible motor effects at low to moderate levels of exposure have not been evaluated.Prenatal exposure to CPF was measured in umbilical cord blood in a sample of 263 inner-city minority children, who were followed prospectively. At approximately 11 years of age (mean age 10.9 ± 0.85 years, range=9.0-13.9), during a neuropsychological assessment, children were asked to draw Archimedes spirals. These were rated by a senior neurologist specializing in movement disorders who was blind to CPF exposure level.Compared to all other children, those with prenatal CPF exposure in the upper quartile range (n=43) were more likely to exhibit mild or mild to moderate tremor (≥ 1) in either arm (p=0.03), both arms (p=0.02), the dominant arm (p=0.01), and the non-dominant arm (p=0.055). Logistic regression analyses showed significant CPF effects on tremor in both arms, either arm, the dominant arm (p-values0.05), and the non-dominant arm (p=0.06), after adjustment for sex, age at testing, ethnicity, and medication.Prenatal CPF exposure is associated with tremor in middle childhood, which may be a sign of the insecticide's effects on nervous system function.

BackgroundAdequate maternal thyroid function during pregnancy is necessary for normal fetal brain development, making pregnancy a critical window of vulnerability to thyroid disrupting insults. Sodium/iodide symporter (NIS) inhibitors, namely perchlorate, nitrate, and thiocyanate, have been shown individually to competitively inhibit uptake of iodine by the thyroid. Several epidemiologic studies examined the association between these individual exposures and thyroid function. Few studies have examined the effect of this chemical mixture on thyroid function during pregnancyObjectivesWe examined the cross sectional association between urinary perchlorate, thiocyanate and nitrate concentrations and thyroid function among healthy pregnant women living in New York City using weighted quantile sum (WQS) regression.MethodsWe measured thyroid stimulating hormone (TSH) and free thyroxine (FreeT4) in blood samples; perchlorate, thiocyanate, nitrate and iodide in urine samples collected from 284 pregnant women at 12 (±2.8) weeks gestation. We examined associations between urinary analyte concentrations and TSH or FreeT4 using linear regression or WQS adjusting for gestational age, urinary iodide and creatinine.ResultsIndividual analyte concentrations in urine were significantly correlated (Spearman's r 0.4–0.5, p

New results from two novel lipid biomarker–based proxies help to constrain the late Cenozoic surface elevation history of the Eastern Cordillera in the tropical northern Andes of Colombia. Previous well-known studies have suggested rapid latest Miocene–Pliocene (6–3 Ma) uplift on the basis of an abrupt shift in pollen species assemblages within sedimentary basin fill of the elevated Bogota Plateau. From resampling of these original study localities, we provide a revised chronology based on magnetic polarity stratigraphy, and we evaluate paleotemperatures using the MBT′/CBT indices (methylation of branched tetraethers/cyclization of branched tetraethers) and hydrogen isotopic composition (δD) of leaf waxes as two independent proxies of past surface elevation. Reconstructed paleotemperatures from the MBT′/CBT proxy show a more gradual cooling trend from ca. 7.6 Ma to present, consistent with less than 1000 m of elevation gain since latest Miocene–Pliocene time and in agreement with geologic evidence for accelerated shortening and exhumation at this time. The leaf wax isotopic data, on the other hand, lack a systematic trend, potentially due to fractionation changes associated with uplift-induced turnover in floral populations. Such changes could obscure isotopic variations in meteoric water, suggesting that leaf-wax isotopic compositions may not provide a direct proxy for elevation in this particular situation involving uplift in a heterogeneous tropical environment. More promising in this case is the MBT′/CBT proxy, which may offer a suitable alternative to carbonate-based paleoelevation proxies in tropical regions where year-round high precipitation rates prevent formation of soil carbonates.

Summary Paralogous transcription factors (TFs) are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein co-factors or the chromatin environment. Using a computational-experimental framework called iMADS (integrative modeling and analysis of differential specificity), we show that, contrary to previous assumptions, paralogous TFs bind differently to genomic target sites even in vitro. We used iMADS to quantify, model, and analyze specificity differences between 11 TFs from 4 protein families. We found that paralogous TFs have diverged mainly at medium- and low-affinity sites, which are poorly captured by current motif models. We identify sequence and shape features differentially preferred by paralogous TFs, and we show that the intrinsic differences in specificity among paralogous TFs contribute to their differential in vivo binding. Thus, our study represents a step forward in deciphering the molecular mechanisms of differential specificity in TF families.

Background: Metal exposure alters neurodevelopmental outcomes; little is known about critical windows of susceptibility when exposure exerts the strongest effect. Objective: To examine associations between dentine biomarkers of manganese (Mn), zinc (Zn) and lead (Pb) and later childhood behaviors. Methods: Subjects enrolled in a longitudinal birth cohort study in Mexico City provided naturally shed deciduous teeth. We estimated weekly prenatal and postnatal dentine Mn, Zn and Pb concentrations in teeth using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and measured behavior at ages 8–11 years of age using the Behavior Assessment System for Children, 2nd edition (BASC-2). We used distributed lag models and lagged weighted quantile sum regression to identify the role of individual and combined dentine biomarkers of Mn, Zn and Pb on behavioral outcomes controlling for maternal education and gestational age. Results: Among the 133 subjects included in this study, prenatal and early postnatal dentine Mn appeared protective against childhood behavioral problems, specifically hyperactivity and attention. Postnatal dentine Mn was associated with increased reporting of internalizing problems, specifically anxiety. At 6 months, a 1-unit increase (unit = 1 SD of log concentration) in Mn was associated with a 0.18-unit (unit = 1 SD of BASC-2 score) increase in internalizing symptoms score and a 0.25-unit increase in anxiety. Postnatal Pb was associated with increasing anxiety symptoms; at 12 months, a 1-unit increase in Pb was associated with a 0.4 unit increase in anxiety symptoms. When examined as a metal mixture, we observed two potential windows of susceptibility to increased anxiety symptoms: the first window (0–8 months) appeared driven by Mn, the second window (8–12 months) was driven by the metal mixture and dominated by Pb. A 1-unit increase in the mixture index was associated with a 0.7-unit increase in SD of anxiety symptoms. Conclusions: Childhood behaviors may demonstrate postnatal windows of susceptibility to individual and mixed metal concentrations measured in deciduous teeth. Prenatal dentine Mn may be protective, while excessive early postnatal Mn may increase risk for adverse behaviors. In combination, higher concentrations of Mn, Zn and Pb may have an adverse impact on behavior. Keywords: Dentine biomarkers, Child behavior, Metal mixtures, Anxiety, Manganese

Aprataxin (APTX) is a DNA-adenylate hydrolase that removes 5′-AMP blocking groups from abortive ligation repair intermediates. XRCC1, a multi-domain protein without catalytic activity, interacts with a number of known repair proteins including APTX, modulating and coordinating the various steps of DNA repair. CK2-phosphorylation of XRCC1 is thought to be crucial for its interaction with the FHA domain of APTX. In light of conflicting reports, the importance of XRCC1 phosphorylation and APTX function is not clear. In this study, a phosphorylation mutant of XRCC1 designed to eliminate APTX binding was stably expressed in Xrcc1−/− cells. Analysis of APTX-GFP accumulation at micro-irradiation damage confirmed that phosphorylated XRCC1 is required for APTX recruitment. APTX-mediated DNA deadenylation activity (i.e., 5′-AMP removal) was measured in extracts of cells expressing wild-type XRCC1 or the XRCC1 phosphorylation mutant, and compared with activity in APTX-deficient and APTX-complemented human cells. APTX activity was lower in extracts from Xrcc1−/− and XRCC1 phosphorylation mutant cells compared to the robust activity in extract from wild-type XRCC1 expressing cells. Taken together, results verify that interaction with phosphorylated XRCC1 is a requirement for significant APTX recruitment to cellular DNA damage and enzymatic activity in cell extracts.

Mitochondrial genome integrity is fundamental to mammalian cell viability. Yet, mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production. DNA repair is important in removing oxidatively‐induced base lesions in mitochondrial DNA, but the presence of a robust base excision repair system has remained unclear. Therefore, we addressed the questions of the presence and repair role of DNA polymerase β (pol β) in mitochondria of mammalian cells. Pol β was localized to mitochondria by electron microscopic immunogold staining and biochemical experiments. Extracts from mitochondria exhibited a strong base excision repair activity that was dependent on pol β. Mitochondria in pol β‐deficient mouse fibroblasts exhibited altered morphology by electron microscopy and were deficient in energy metabolism. These results indicate mammalian mitochondria have a robust base lesion repair system.

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1(+/+) and Xrcc1(-/-) mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.

Members of transcription factor (TF) families, i.e. paralogous TFs, are oftentimes reported to have identical DNA-binding motifs, despite the fact that they perform distinct regulatory functions in the cell. Differential genomic targeting by paralogous TFs is generally assumed to be due to interactions with protein cofactors or the chromatin environment. Contrary to previous assumptions, we find that paralogous TFs have different intrinsic preferences for DNA, not captured by current motif models, and these differences partly explain differential genomic binding and functional specificity. Our finding was possible due to a unique combination of carefully designed high-throughput assays and rigorous computation modeling, integrated into a unified framework called iMADS. We used iMADS to quantify, model, and analyze specificity differences between 11 paralogous TFs from 4 distinct human TF families. Our finding of differential specificity between closely related TFs has important implications for the interpretation of the regulatory effects of non-coding genetic variants.

Purpose In diabetic retinopathy (DR) microvascular damage results from ischaemia driven production of pro-angiogenic vascular endothelial growth factor (VEGF) inducing angiogenesis and increased permeability in the retina. Small molecule inhibitors of serine-rich protein kinase-1 (SRPK1), with good penetration properties to the retina, have been shown to inhibit choroidal neovascularisation in mice as eye drops by decreasing pro-angiogenic and increasing anti-angiogenic VEGF isoforms. SRPK1 inhibitors such as SPHINX31 may therefore switch splicing in DR and prevent increased vascular permeability. Methods FFA was performed in Norway Brown rats on day 0 and 7, using the Micron IV retinal microscope (Phoenix Research). Animals received twice daily topical eye drops with eye formulation only control buffer (n = 5) or SPHINX31 (200 μg/ml, n = 6). On day 1 animals received a single dose of streptozotocin (50 mg/kg, i.p.) to induce type I diabetes and maintained for 1 week without insulin. The ratio of interstitial to vascular fluorescence was calculated and plotted against time to determine an estimate of permeability. Results Retinal permeability (12.67 ± 1.09 × 10−4 cms−1) was shown to significantly increase (p

Repair of DNA-protein crosslinks and oxidatively damaged DNA base lesions generates intermediates with nicks or gaps with abnormal and blocked 3'-phosphate and 5'-OH ends that prevent the activity of DNA polymerases and ligases. End cleaning in mammalian cells by Tdp1 and PNKP produces the conventional 3'-OH and 5'-phosphate DNA ends suitable for completion of repair. This repair function of PNKP is facilitated by its binding to the scaffold protein XRCC1, and phosphorylation of XRCC1 by CK2 at several consensus sites enables PNKP binding and recruitment to DNA damage. To evaluate this documented repair process, a phosphorylation mutant of XRCC1, designed to eliminate PNKP binding, was stably expressed in Xrcc1-/- mouse fibroblast cells. Analysis of PNKP-GFP accumulation at micro-irradiation induced damage confirmed that the XRCC1 phosphorylation mutant failed to support efficient PNKP recruitment, whereas there was rapid recruitment in cells expressing wild-type XRCC1. Recruitment of additional fluorescently-tagged repair factors PARP-1-YFP, GFF-XRCC1, PNKP-GFP and Tdp1-GFP to micro-irradiation induced damage was assessed in wild-type XRCC1-expressing cells. PARP-1-YFP recruitment was best fit to two exponentials, whereas kinetics for the other proteins were fit to a single exponential. The similar half-times of recruitment suggest that XRCC1 may be recruited with other proteins possibly as a pre-formed complex. Xrcc1-/- cells are hypersensitive to the DNA-protein cross-link inducing agent camptothecin (CPT) and the DNA oxidative agent H2O2 due in part to compromised PNKP-mediated repair. However, cells expressing the PNKP interaction mutant of XRCC1 demonstrated marked reversal of CPT hypersensitivity. This reversal represents XRCC1-dependent repair in the absence of the phosphorylation-dependent PNKP recruitment and suggests either an XRCC1-independent mechanism of PNKP recruitment or a functional back-up pathway for cleaning of blocked DNA ends.

Fluorescently-tagged repair proteins have been widely used to probe recruitment to micro-irradiation-induced nuclear DNA damage in living cells. Here, we quantify APE1 dynamics after micro-irradiation. Markers of DNA damage are characterized and UV-A laser micro-irradiation energy conditions are selected for formation of oxidatively-induced DNA base damage and single strand breaks, but without detectable double strand breaks. Increased energy of laser micro-irradiation, compared with that used previously in our work, enables study of APE1 dynamics at the lesion site. APE1 shows rapid transient kinetics, with recruitment half-time of less than 1 s and dissociation half-time of less than 15 s. In cells co-transfected with APE1 and PARP1, the recruitment half-time of PARP1 was slower than that of APE1, indicating APE1 is a rapid responder to the damage site. While recruitment of APE1 is unchanged in the presence of co-transfected PARP1, APE1 dissociation is 3-fold slower, revealing PARP1 involvement in APE1 dynamics. Further, we find that APE1 dissociation kinetics are strongly modified in the absence of DNA polymerase β (pol β). After unchanged recruitment to the damage site, dissociation of APE1 became undetectable. This indicates a necessary role for pol β in APE1 release after its recruitment to the damage site. These observations represent an advance in our understanding of in vivo dynamics of base excision repair factors APE1, PARP1 and pol β.

PARP-1 is important for the recognition of both endogenous and exogenous DNA damage, and binds to DNA strand breaks including intermediates of base excision repair (BER). Once DNA-bound, PARP-1 becomes catalytically activated synthesizing PAR polymers onto itself and other repair factors (PARylation). As a result, BER repair proteins such as XRCC1 and DNA polymerase β (pol β) are more efficiently and rapidly recruited to sites of DNA damage. In the presence of an inhibitor of PARP activity (PARPi), PARP-1 binds to sites of DNA damage, but PARylation is prevented. BER enzyme recruitment is hindered, but binding of PARP-1 to DNA is stabilized, impeding DNA repair and leading to double-strand DNA breaks (DSB). Deficiencies in pol β−/− and Xrcc1−/− cells resulted in hypersensitivity to the PARP inhibitor 4-AN and reexpression of pol β or XRCC1, in these contexts, reversed the 4-AN hypersensitivity phenotype. BER deficiencies also showed evidence of replication defects that lead to DSB-induced apoptosis upon PARPi treatment. Finally, the clinically relevant PARP inhibitors olaparib and veliparib also exhibited hypersensitivity in both pol β−/− and Xrcc1−/− BER-deficient cells. These results reveal heightened sensitivity to PARPi as a function of BER deficiency. Implications: BER deficiency represents a new therapeutic opportunity to enhance PARPi efficacy. Visual Overview: http://mcr.aacrjournals.org/content/12/8/1128/F1.large.jpg. Mol Cancer Res; 12(8); 1128–39. ©2014 AACR.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase β (pol β) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol β in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol β is found in vivo, and it is known that pol β and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol β, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol β (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol β in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides., Oxidative stress in cells leads to the oxidations of DNA precursors. Here the authors show that these oxidized precursors can be incorporated in vivo during base excision repair, leading to DNA breaks and cytotoxicity.

The multi-domain protein XRCC1 is without catalytic activity, but can interact with a number of known repair proteins. The interaction between the N-terminal domain (NTD) of XRCC1 and DNA polymerase β (pol β) is critical for recruitment of pol β to sites of DNA damage and repair. Crystallographic and NMR approaches have identified oxidized and reduced forms of the XRCC1 NTD, and the corresponding forms of XRCC1 have been identified in cultured mouse fibroblast cells. Both forms of NTD interact with pol β, but the interaction is much stronger with the oxidized form. The potential for formation of the C12-C20 oxidized conformation can be removed by alanine substitution at C12 (C12A) leading to stabilized reduced XRCC1 with a lower pol β binding affinity. Here, we compare cells expressing C12A XRCC1 (XRE8) with those expressing wild-type XRCC1 (XC5). Reduced C12A XRCC1 is detected at sites of micro-irradiation DNA damage, but provides slower recruitment of pol β. Expression of reduced XRCC1 does not affect sensitivity to MMS or H2O2. In contrast, further oxidative stress imposed by glutathione depletion results in increased sensitization of reduced XRCC1-expressing cells to H2O2 compared with wild-type XRCC1-expressing cells. There is no indication of enhanced H2O2-generated free radicals or DNA strand breaks in XRE8 cells. However, elevated cellular PAR is found following H2O2 exposure, suggesting BER deficiency of H2O2-induced damage in the C12A expressing cells.

Prenatal exposure to chlorpyrifos (CPF), an organophosphate insecticide, is associated with neurobehavioral deficits in humans and animal models. We investigated associations between CPF exposure and brain morphology using magnetic resonance imaging in 40 children, 5.9–11.2 y, selected from a nonclinical, representative community-based cohort. Twenty high-exposure children (upper tertile of CPF concentrations in umbilical cord blood) were compared with 20 low-exposure children on cortical surface features; all participants had minimal prenatal exposure to environmental tobacco smoke and polycyclic aromatic hydrocarbons. High CPF exposure was associated with enlargement of superior temporal, posterior middle temporal, and inferior postcentral gyri bilaterally, and enlarged superior frontal gyrus, gyrus rectus, cuneus, and precuneus along the mesial wall of the right hemisphere. Group differences were derived from exposure effects on underlying white matter. A significant exposure × IQ interaction was derived from CPF disruption of normal IQ associations with surface measures in low-exposure children. In preliminary analyses, high-exposure children did not show expected sex differences in the right inferior parietal lobule and superior marginal gyrus, and displayed reversal of sex differences in the right mesial superior frontal gyrus, consistent with disruption by CPF of normal behavioral sexual dimorphisms reported in animal models. High-exposure children also showed frontal and parietal cortical thinning, and an inverse dose–response relationship between CPF and cortical thickness. This study reports significant associations of prenatal exposure to a widely used environmental neurotoxicant, at standard use levels, with structural changes in the developing human brain.

Background: In a longitudinal birth cohort study of inner-city mothers and children (Columbia Center for Children’s Environmental Health), we have previously reported that prenatal exposure to chlorpyrifos (CPF) was associated with neurodevelopmental problems at 3 years of age. Objective: The goal of the study was to estimate the relationship between prenatal CPF exposure and neurodevelopment among cohort children at 7 years of age. Methods: In a sample of 265 children, participants in a prospective study of air pollution, we measured prenatal CPF exposure using umbilical cord blood plasma (picograms/gram plasma) and 7-year neurodevelopment using the Wechsler Intelligence Scale for Children, 4th edition (WISC-IV). Linear regression models were used to estimate associations, with covariate selection based on two alternate approaches. Results: On average, for each standard deviation increase in CPF exposure (4.61 pg/g), Full-Scale intelligence quotient (IQ) declined by 1.4% and Working Memory declined by 2.8%. Final covariates included maternal educational level, maternal IQ, and quality of the home environment. We found no significant interactions between CPF and any covariates, including the other chemical exposures measured during the prenatal period (environmental tobacco smoke and polycyclic aromatic hydrocarbons). Conclusions: We report evidence of deficits in Working Memory Index and Full-Scale IQ as a function of prenatal CPF exposure at 7 years of age. These findings are important in light of continued widespread use of CPF in agricultural settings and possible longer-term educational implications of early cognitive deficits.

OBJECTIVE: Recent pesticide-monitoring results suggest that a shift in residential pesticide exposure from organophosphorus insecticides to pyrethroid insecticides has occurred. Pyrethroid insecticides are potential neurodevelopmental toxicants and have not been evaluated for developmental toxicity. Our objective was to explore the association between prenatal exposure to permethrin (a common pyrethroid) and piperonyl butoxide (a pyrethroid synergist) and 36-month neurodevelopment. METHODS: Participants is this study were part of a prospective cohort of black and Dominican mothers and newborns living in low-income neighborhoods in New York City. We examined 36-month cognitive and motor development (using the Bayley Scales of Infant Development, second edition) as a function of permethrin levels measured in maternal and umbilical cord plasma collected on delivery and permethrin and piperonyl butoxide levels measured in personal air collected during pregnancy. All models were controlled for gender, gestational age, ethnicity, maternal education, maternal intelligence, quality of the home environment, and prenatal exposure to environmental tobacco smoke and chlorpyrifos. RESULTS: Prenatal exposure to permethrin in personal air and/or plasma was not associated with performance scores for the Bayley Mental Developmental Index or the Psychomotor Developmental Index. After data adjustment, children more highly exposed to piperonyl butoxide in personal air samples (>4.34 ng/m3) scored 3.9 points lower on the Mental Developmental Index than those with lower exposures (95% confidence interval: −0.25 to −7.49). CONCLUSIONS: Prenatal exposure to piperonyl butoxide was negatively associated with 36-month neurodevelopment.

The combination of poly(ADP-ribose)polymerase (PARP) inhibitors and alkylating agents is currently being investigated in cancer therapy clinical trials. However, the DNA lesions producing the synergistic cell killing effect in tumors are not fully understood. Treatment of human and mouse fibroblasts with the monofunctional DNA methylating agent methyl methanesulfonate (MMS) in the presence of a PARP inhibitor has been shown to trigger a cell cycle checkpoint response. Among other changes, this DNA damage response to combination treatment includes activation of ATM/Chk2 and phosphorylation of histone H2A.X. These changes are consistent with DNA double-strand break (DSB) formation during the response, but the measurement of DSBs has not been addressed. Such DSB evaluation is important in understanding this DNA damage response because events other than DSB formation are known to lead to ATM/Chk2 activation and H2A.X phosphorylation. Here, we examined the structural integrity of genomic DNA after the combined treatment of cells with MMS and a PARP inhibitor, i.e., exposure to a sub-lethal dose of MMS in the presence of the PARP inhibitor 4-amino-1,8-napthalimide (4-AN). We used pulsed field gel electrophoresis (PFGE) for measurement of DSBs in both human and mouse embryonic fibroblasts, and flow cytometry to follow the phosphorylated form of H2A.X (gamma-H2A.X). The results indicate that DSBs are formed with the combination treatment, but not following treatment with either agent alone. Our data also show that formation of gamma-H2A.X correlates with PARP-1-expressing cells in S-phase of the cell cycle. The observations support the model that persistence of PARP-1 at base excision repair intermediates, as cells move into S-phase, leads to DSBs and the attendant checkpoint responses.

Environmental contaminants pose a threat to infant neurodevelopment. In this current paper, we discuss evidence for the potentially harmful impact of fetal and early childhood exposure to polycyclic aromatic hydrocarbons (PAHs), environmental tobacco smoke (ETS), and organophosphorus (OP) insecticides. We focus on effects resulting from chronic and low-level exposure during the prenatal period and early childhood, when the brain is still undergoing rapid developmental changes.

The Columbia Center for Children's Environmental Health (CCCEH) previously reported widespread residential insecticide use in urban communities in New York City. Research suggests that pyrethroids are replacing organophosphates (OPs) in response to 2000-2001 US EPA pesticide regulations restricting OP use. A systematic assessment of active ingredients used for residential pest control is lacking. We queried a database of pesticide applications reported by licensed applicators between 1999 and 2005 and surveyed pest control products available in 145 stores within 29 zip codes in the CCCEH catchment area including Northern Manhattan and the South Bronx. Pyrethroids, pyrethrins, piperonyl butoxide, and hydramethylnon were the most common insecticide active ingredients reported as used by licensed pesticide applicators within the 29 zip codes of the CCCEH catchment area between 1999 and 2005. Use of certain pyrethroids and some non-spray insecticides such as fipronil and boric acid increased significantly by year (logistic regression, OR>1.0, P

To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14 h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair capacity for uracil-DNA in wild type mouse fibroblasts was very strong, whereas the repair capacity for THF-DNA, although strong, was slightly weaker. Repair capacity in DNA polymerase beta (Pol beta) null cells for uracil-DNA and THF-DNA was reduced by approximately 15% and 20%, respectively, compared to that in wild type cells. In both cases, the repair deficiency was fully complemented in Pol beta null cells expressing recombinant Pol beta. The effect of inhibition of poly(ADP-ribose) polymerase (PARP) activity on repair capacity was examined by treatment of cells with the inhibitor 4-amino-1,8-naphthalimide (4-AN). PARP inhibition decreased the repair capacity for both lesions in wild type cells, and this reduction was to the same level as that seen in Pol beta null cells. In contrast, 4-AN had no effect on repair in Pol beta null cells. The results highlight that Pol beta and PARP function in the same repair pathway, but also suggest that there is repair independent of both Pol beta and PARP activities. Thus, before the BER capacity of a cell can be predicted or modulated, a better understanding of Pol beta and PARP activity-independent BER pathways is required.

Dear Editor DNA repair plays a major role in maintaining genomic integrity such that deficiency, as well as an excess of DNA repair, has a significant impact. For example, the absence of DNA polymerase β(pol β) expression in mice results in embryonic lethality, while over-expression of pol β causes cataracts [1,2]. Alkylated DNA bases in higher eukaryotes are repaired predominantly by the base excision repair (BER) pathway. During “single-nucleotide” BER, an alkylated base is removed by the damage specific glycosylase, 3-methyladenine DNA glycosylase, and the resulting abasic site is cleaved by apurinic/apyrimidinic endonuclease. This results in a nick with a 5’ dRP group that can be removed by the dRP lyase activity of pol β; the same polymerase then fills the single-nucleotide gap. Finally, a DNA ligase seals the nick completing repair (reviewed in [3]). Mouse embryonic fibroblasts (MEFs) deficient in pol β demonstrate increased sensitivity to alkylating agents such as methyl methanesulfonate (MMS) [4]. Hypersensitivity is moderate, and cellular extracts from pol β null cells have some residual BER activity, suggesting that other polymerases could participate in BER. Reversal of the MMS hypersensitivity requires the dRP lyase activity of pol β [5]. DNA polymerase λ(pol λ) also has dRP lyase activity and can function as a back up BER enzyme in pol β null cells [6,7]. Another DNA polymerase carrying dRP lyase activity is DNA polymerase ι(pol ι) [7,8]. This polymerase is a member of the Y-family of DNA polymerases and was identified due to its homology with DNA polymerase η(pol η) [9]. Based on in vitro biochemical data and on the structure of the enzyme, it has been proposed that pol ι participates in DNA translesion synthesis during bypass of a damaged template [10,11]. Gene expression at the mRNA level is modest in most tissues, with strongest expression in testis and ovarian cells [9]. The data on the biological roles of this enzyme are contradictory. Pol ι is expressed at a high level in some human cancer cells and also is associated with increased frequency of mutagenesis in certain cell types; lowering the expression of this enzyme decreased mutation frequency [12]. Deletion of the pol ι gene in human Burkitt’s lymphoma cells reduced inducible somatic hypermutation (SHM), and this process could be restored by expression of pol ι [13,14]. At the same time, a mouse strain deficient in expression of pol ι demonstrated the same ability for SHM as mice carrying the wild-type pol ι gene [15,16]. Down-regulation of human pol ι in HEK 293 cells showed no effect on resistance to UV-induced damage as well as to UVC-induced mutagenesis [17]. Recent studies have revealed that MEFs derived from the 129 mouse strain carry a genomic mutation causing premature termination of the pol ι transcript and preventing expression of active enzyme [16]. Therefore, we analyzed MEF lines developed in this laboratory, and that are deficient and proficient in pol β, for expression of pol ι. Utilizing RT-PCR, sequencing and immunoblotting, we confirmed that pol β-deficient Mβ19tsA MEFs are also deficient in expression of pol ι (Fig. 1 and data not shown). This is essentially in agreement with previously published data from another laboratory [18]. Since MEF cells deficient in BER are hypersensitive to alkylating agent-induced DNA damage, we next determined the resistance of MEFs to the monofunctional alkylating agent MMS. This agent methylates predominantly adenine and guanine bases producing mono-adducts that block DNA replication [19] and are removed from DNA primarily by glycosylase-initiated pol β-dependent BER [5]. Fig. 1 Expression of pol ι in MEFs. Western blotting of whole cell extract (20 µg) isolated from Mβ16tsA (lane 1), Mβ19tsA (lane 2), 92TAg (lane 3), and 88TAg (lane 4) cell lines. Cell lines were grown in DMEM medium supplemented ... To identify a role for pol ι in MEF cellular resistance to MMS in a pol β null background, we used Mβ19tsA cells that lack expression of both pol ι and pol β (Fig. 1). As a control, we used Mβ16tsA and 92TAg cells that are pol β and pol ι proficient, and 88TAg cells deficient in pol β and proficient in pol ι expression (Fig. 1). Treatment with MMS resulted in growth inhibition of cells in a dose dependent manner (Fig. 2A) as was shown previously [4,20]. MMS had relatively less effect on wild-type Mβ16tsA and 92TAg cell lines, especially at lower concentrations, whereas Mβ19tsA and 88TAg pol β null cell lines showed clear hypersensitivity to MMS (Fig. 2A). While the LD10 doses for wild-type Mβ16tsA and 92TAg were indistinguishable (~2.2 mM MMS), the LD10 for Mβ19tsA and 88TAg were dissimilar (0.75 and 1.25 mM, respectively). Therefore, pol β deficient cells expressing pol ι (88TAg) showed increased resistance to MMS compared with cells that were additionally deficient in pol ι (Mβ19tsA) (Fig. 2A). Fig. 2 Growth inhibition assay after exposure to DNA damaging agents. (A) 92TAg (closed squares), 88TAg (open squares), Mβ16tsA (closed circles) and Mβ19tsA (open circles) were treated with MMS for 1 h. (B) Mβ19tsA (open circles) and ... The pol β-deficient MEFs used in this study had different origins. To minimize the effect of genetic background variability, we expressed mouse pol ι in Mβ19tsA pol ι- and pol β-deficient cells at a level equal to, and greater than, the wild-type level. Analysis of growth inhibition revealed that expression of pol ι did not affect the resistance of these cells to the alkylating agents MMS and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (Fig. 2B and C). Thus, we conclude that the difference in MMS sensitivity of the pol β-deficient Mβ19tsA and 88TAg cell lines is not likely to be a result of the difference in pol ι expression. Finally, since it has been proposed that pol ι participates in error-prone bypass across thymine dimmers, we tested the sensitivity of cells expressing pol ι to UV radiation. While the individual clones have slight variations, we could not find any statistically significant differences between cells expressing pol ι and those expressing control vector (Fig. 2D). There are at least 15 DNA polymerases currently described in mammalian cells [21]. Three of these polymerases, pol α, pol γ, and pol e are indispensable, while others showed various effects on MEF cell viability. The functional interactions or overlap between these enzymes are of special interest. Thus, the polymerases in higher organisms appear to either substitute for each other or participate in similar processes. Our results demonstrate that expression of pol ι has negligible impact on the alkylation damage sensitivity of cells lacking pol β.

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase beta (pol beta) is specific to this pathway, whereas pol beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.

Carbonate oxygen isotope paleoaltimetry is based on analysis of the d 18 O value of carbonate precipitated from surface water. Deciphering the diagenetic history is important for establishing whether particular carbonates are accurate recorders of paleosurface waters, which reflect paleoelevation. This study provides examples from southern, east–central, and northeastern Tibet of approaches aimed at evaluating the diagenetic history of lacustrine micrites and pedogenic carbonates. The most desirable technique for avoiding erroneous interpretations related to diagenetic overprinting is to analyze carbonates that are known to be primary, such as aragonitic shell material. In rocks that do not contain shell material, we have compared lacustrine micrites and pedogenic carbonates to diagenetic carbonate phases to determine the effects of diagenesis on the isotopic composition of primary carbonates. Where the potential effects of diagenesis are subtle or ambiguous, we have evaluated the fidelity of the carbonate record from systematic trends in C and O isotopes that agree with other interpretations of paleoenvironment, such as high frequency covariance in C and O that corresponds with changes in the Mg concentration of carbonates. Using these strategies, we have determined that diagenesis has not affected the isotopic composition of carbonates in the Late Miocene–Pliocene Thakkhola graben in southern Tibet and the Oligocene to Pliocene Linxia basin in northeastern Tibet. In the case of Paleogene basins in east–central Tibet, however, ambiguity in data precludes the determination of diagenetic effects. D 2004 Elsevier B.V. All rights reserved.